Intracellular glycation of nuclear DNA, mitochondrial DNA, and cytosolic proteins triggered by endogenous oxidative stress [Elektronische Ressource] = Intrazelluläre Glykierung genomischer DNA, mitochondrialer DNA und zytosolischer Proteine ausgelöst durch endogenen oxidativen Stress / vorgelegt von Viola Breyer

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Intracellular glycation of nuclear DNA,mitochondrial DNA, and cytosolic proteinstriggered by endogenous oxidative stressIntrazelluläre Glykierung genomischer DNA,mitochondrialer DNA und zytosolischer Proteineausgelöst durch endogenen oxidativen StressDer Naturwissenschaftlichen Fakultät derFriedrich-Alexander-Universität Erlangen-Nürnbergzur Erlangung des DoktorgradesDr. rer. nat.vorgelegt vonViola Breyeraus DachauAls Dissertation genehmigt von derNaturwissenschaftlichen Fakultät derFriedrich-Alexander-Universität Erlangen-NürnbergTag der mündlichen Prüfung: 4. März 2011Vorsitzender der Prüfungskommission: Prof. Dr. Rainer FinkErstberichterstatterin: Prof. Dr. Monika PischetsriederZweitberichterstatterin: Prof. Dr. Ting-Ting HuangParts of this thesis have already been published:PublicationsBreyer, V., Frischmann, M., Bidmon, C., Schemm, A., Schiebel, K., Pischetsrieder, M.Analysis and biological relevance of advanced glycation end-products of DNA in eukaryoticcells.Febs J, 275(5):914–25, 2008Breyer, V., Pischetsrieder, M.Simultaneous analysis of advanced glycation end-products of nuclear DNA, mitochondrialDNA, and cytosolic proteins.submittedTalksBreyer, V., Pischetsrieder, M.Non-Enzymatic Modifications of Proteins and DNA During Senescence3rd EuCheMS Chemistry Congress, Nuremberg, Germany, 2010Breyer, V., Pischetsrieder, M.
Publié le : samedi 1 janvier 2011
Lecture(s) : 88
Source : D-NB.INFO/1011059290/34
Nombre de pages : 171
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Intracellular glycation of nuclear DNA,
mitochondrial DNA, and cytosolic proteins
triggered by endogenous oxidative stress
Intrazelluläre Glykierung genomischer DNA,
mitochondrialer DNA und zytosolischer Proteine
ausgelöst durch endogenen oxidativen Stress
Der Naturwissenschaftlichen Fakultät der
Friedrich-Alexander-Universität Erlangen-Nürnberg
zur Erlangung des Doktorgrades
Dr. rer. nat.
vorgelegt von
Viola Breyer
aus DachauAls Dissertation genehmigt von der
Naturwissenschaftlichen Fakultät der
Friedrich-Alexander-Universität Erlangen-Nürnberg
Tag der mündlichen Prüfung: 4. März 2011
Vorsitzender der Prüfungskommission: Prof. Dr. Rainer Fink
Erstberichterstatterin: Prof. Dr. Monika Pischetsrieder
Zweitberichterstatterin: Prof. Dr. Ting-Ting HuangParts of this thesis have already been published:
Publications
Breyer, V., Frischmann, M., Bidmon, C., Schemm, A., Schiebel, K., Pischetsrieder, M.
Analysis and biological relevance of advanced glycation end-products of DNA in eukaryotic
cells.
Febs J, 275(5):914–25, 2008
Breyer, V., Pischetsrieder, M.
Simultaneous analysis of advanced glycation end-products of nuclear DNA, mitochondrial
DNA, and cytosolic proteins.
submitted
Talks
Breyer, V., Pischetsrieder, M.
Non-Enzymatic Modifications of Proteins and DNA During Senescence
3rd EuCheMS Chemistry Congress, Nuremberg, Germany, 2010
Breyer, V., Pischetsrieder, M.
Simultaneous Analysis of AGEs of Genomic DNA, Mitochondrial DNA, and Cytosolic
Proteins in a Cellular Model for Aging
10th International Symposium on the Maillard Reaction, Palm Cove, Australia, 2009
Poster
Breyer, V., Pischetsrieder, M.
Influence of Oxidative Stress on the Formation of Advanced Glycation End-Products
16th Annual Meeting of the Society of Free Radicals in Biology and Medicine, San
Francisco, U.S.A., 2009CONTENTS
Abstract 1
Zusammenfassung 7
1 Introduction 13
1.1 The free radical theory of aging . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.2 The mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.2.1 Citric acid cycle and fatty acid-oxidation . . . . . . . . . . . . . 16
1.2.2 Oxidative phosphorylation . . . . . . . . . . . . . . . . . . . . . . . 16
1.2.3 The mitochondrial role in aging . . . . . . . . . . . . . . . . . . . . 18
1.3 Reactive oxygen species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
1.4 Advanced glycation end-products . . . . . . . . . . . . . . . . . . . . . . . . 21
1.4.1 The Maillard reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
1.4.2 Physiological consequences . . . . . . . . . . . . . . . . . . . . . . . 21
1.4.3 The glycation theory of aging . . . . . . . . . . . . . . . . . . . . . 28
1.5 Mouse models with reduced SOD activity . . . . . . . . . . . . . . . . . . . 29
1.5.1 Mutant mice lacking copper/zinc superoxide dismutase . . . . . 30
1.5.2 Mutant mice lacking manganese . . . . . . 31
1.6 Aims of this thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2 In vitro analysis of DNA glycation products 35
2.1 Introduction and experimental design . . . . . . . . . . . . . . . . . . . . 35
2.1.1 Selection of a reporter gene assay . . . . . . . . . . . . . . . . . . 36
2.1.2 of the transfection method . . . . . . . . . . . . . . . . . . 37
2.1.3 Introduction and quantification of CEdG modifications . . . . . 38ii CONTENTS
2.1.4 Influence of CEdG adducts on luciferase activity in HEK 293 T
cells in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
2.2.1 Cytotoxic effect on transfected HEK 293 T cells . . . . . . . . . . . 42
2.2.2 Quantification of plasmid transcription . . . . . . . . . . . . . . . . 42
2.2.3 Influence of CEdG adducts on the functionality of the ampicillin
resistance gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.2.4 Restriction enzyme digestion of CEdG-modified plasmid DNA . 46
2.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3 Simultaneous analysis of endogenously formed AGEs 51
3.1 Introduction and experimental design . . . . . . . . . . . . . . . . . . . . . 51
3.1.1 In vitro aging model . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
3.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.2.1 Development of a method for the simultaneous isolation of
nuclear DNA, mtDNA, and cytosolic proteins . . . . . . . . . . . . 55
3.2.2 Isolation of nuclear DNA, mitochondrial DNA, and cytosolic
proteins from NIH 3T3 fibroblasts . . . . . . . . . . . . . . . . . . . 59
3.2.3 Characterization of the mitochondrial fraction . . . . . . . . . . . . 59
3.2.4 Determination of optimal conditions for the senescence-like
growth arrest of NIH 3T3 fibroblasts . . . . . . . . . . . . . . . . . . 61
3.2.5 of CEdG content of nuclear DNA and mtDNA . . . 64
3.2.6 Analysis of protein expression and AGE modifications in senes-
cent fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.2.7 Analysis of oxidative protein modification . . . . . . . . . . . . . . 69
3.2.8 Identification of oxidized proteins . . . . . . . . . . . . . . . . . . . 71
3.2.9 Immunoprecipitation of actin and subsequent analysis for oxida-
tive modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
3.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
4 Formation of AGEs in SOD knockout mice 79
4.1 Introduction and experimental design . . . . . . . . . . . . . . . . . . . . . 79
4.1.1 Antioxidant status in SOD mutant mice . . . . . . . . . . . . . . . . 81
4.1.2 Fetal fibroblasts from MnSOD mutant mice . . . . . . . . . . . . . . 82
4.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
4.2.1 In vitro modification of DNA by oxidative stress . . . . . . . . . . 83CONTENTS iii
4.2.2 Preparation of primary MEFs . . . . . . . . . . . . . . . . . . . . . 83
+=+ =+4.2.3 Culturing Sod2 and Sod2 MEFs . . . . . . . . . . . . . . . . . 84
4.2.4 Development of a method for the simultaneous isolation of
nuclear DNA, mtDNA, and cytosolic proteins from primary MEFs 86
4.2.5 Isolation of nuclear DNA, mtDNA and cytosolic proteins from
+=+ =+Sod2 and Sod2 fibroblasts . . . . . . . . . . . . . . . . . . . . 87
4.2.6 Characterization of the mitochondrial fraction . . . . . . . . . . . . 89
4.2.7 Determination of CEdG content of nuclear DNA and mtDNA . . . 89
4.2.8 Analysis of protein expression and AGE modifications . . . . . . 90
4.2.9 of oxidative protein expression . . . . . . . . . . . . . . . . 91
4.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
5 Materials and methods 97
5.1 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
5.2 Laboratory equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
5.3 Chemicals and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
5.4 Methods for the in vitro analysis of DNA glycation products . . . . . . . . 109
5.4.1 Cultivation of HEK 293 T cells . . . . . . . . . . . . . . . . . . . . . . 109
5.4.2 Plasmid DNA preparation . . . . . . . . . . . . . . . . . . . . . . . 110
5.4.3 Introduction of CEdG modifications . . . . . . . . . . . . . . . . . 110
5.4.4 Competitive ELISA for CEdG . . . . . . . . . . . . . . . . . . . . . . . 112
5.4.5 Transient transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
5.4.6 Reporter gene assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
5.4.7 Cytotoxic effect on transfected HEK 293 T cells . . . . . . . . . . 113
5.4.8 Transformation of glycated DNA to a bacterial host . . . . . . . . 113
5.4.9 Restriction digestion of CEdG-modified plasmids . . . . . . . . . 113
5.4.10 RNA isolation and RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . 114
5.5 Methods for the analysis of endogenously formed AGEs in NIH 3T3
fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
5.5.1 Cultivation of NIH 3T3 mouse embryo fibroblasts . . . . . . . . . . 114
5.5.2 Freezing and thawing of NIH 3T3 fibroblasts . . . . . . . . . . . . 115
5.5.3 Simultaneous isolation of nuclei, mitochondria, and cytosolic
proteins from NIH 3T3 fibroblasts . . . . . . . . . . . . . . . . . . 115
5.5.4 Isolation of DNA from nuclei and mitochondria . . . . . . . . . . 116
5.5.5 Analysis of cell proliferation . . . . . . . . . . . . . . . . . . . . . . 116
5.5.6 of cell viability . . . . . . . . . . . . . . . . . . . . . . . . . . 117iv CONTENTS
5.5.7 Senescence-like growth arrest of NIH 3T3 fibroblasts . . . . . . . 117
5.5.8 Assay for succinate dehydrogenase activity . . . . . . . . . . . . . 118
5.5.9 PCR analysis of the isolated mtDNA . . . . . . . . . . . . . . . . . 118
5.5.10 Competitive ELISA for CEdG . . . . . . . . . . . . . . . . . . . . . . 118
5.5.11 Quantification of protein yield . . . . . . . . . . . . . . . . . . . . . . 119
5.5.12 Gel-electrophoretic separation of cytosolic proteins . . . . . . . . . 119
5.5.13 2-D gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . 120
5.5.14 AGE-CML ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
5.5.15 Ultrafiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
5.5.16 Synthesis of CML-modified and CEL-modified BSA . . . . . . . . 123
5.5.17 Test for CML-antibody specificity . . . . . . . . . . . . . . . . . . . 123
5.5.18 Western blot analysis for AGE-modified proteins . . . . . . . . . . 124
5.5.19 Western blot for actin . . . . . . . . . . . . . . . . . . . . . . . . . . 125
5.5.20 Western blot analysis for oxidative protein modifications . . . . 126
5.5.21 Partial enzymatic protein hydrolysis in gel . . . . . . . . . . . . . 126
5.5.22 MALDI-TOF-MS analysis . . . . . . . . . . . . . . . . . . . . . . . . . 127
5.5.23 Immunoprecipitation of actin . . . . . . . . . . . . . . . . . . . . . . 127
5.6 Methods for the analysis of AGEs in nuclear DNA, mtDNA, and cytosolic
proteins of SOD2 MEFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
5.6.1 In vitro incubation of DNA with hydrogen peroxide . . . . . . . 128
5.6.2 Breeding and genotyping . . . . . . . . . . . . . . . . . . . . . . . . . 129
5.6.3 Preparation of primary mouse embryonic fibroblasts (MEFs) . . . 129
5.6.4 Cultivation of MEFs . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
5.6.5 Growth curves of MEFs . . . . . . . . . . . . . . . . . . . . . . . . . 130
5.6.6 Simultaneous isolation of nuclei, mitochondria, and cytosolic
proteins from MEFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
5.6.7 Isolation of DNA from nuclei and mitochondria . . . . . . . . . . . 132
5.6.8 Assay for succinate dehydrogenase activity . . . . . . . . . . . . . . 132
5.6.9 Competitive ELISA for CEdG . . . . . . . . . . . . . . . . . . . . . . 133
5.6.10 Western blot analysis for AGE-modified proteins . . . . . . . . . 133
5.6.11 Western blot for oxidative protein modifications . . . . 133
5.7 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Bibliography 135
List of abbreviations 153CONTENTS v
List of figures 157
List of tables 159
Danksagung 161

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